Defining the Kv2.1-syntaxin molecular interaction identifies a first-in-class small molecule neuroprotectant.

The neuronal cell death-promoting loss of cytoplasmic K+ following injury is mediated by an increase in Kv2.1 potassium channels in the plasma membrane. This phenomenon relies on Kv2.1 binding to syntaxin 1A via 9 amino acids within the channel intrinsically disordered C terminus. Preventing this interaction with a cell and blood-brain barrier-permeant peptide is neuroprotective in an in vivo stroke model. Here a rational approach was applied to define the key molecular interactions between syntaxin and Kv2.1, some of which are shared with mammalian uncoordinated-18 (munc18). Armed with this information, we found a small molecule Kv2.1-syntaxin-binding inhibitor (cpd5) that improves cortical neuron survival by suppressing SNARE-dependent enhancement of Kv2.1-mediated currents following excitotoxic injury. We validated that cpd5 selectively displaces Kv2.1-syntaxin-binding peptides from syntaxin and, at higher concentrations, munc18, but without affecting either synaptic or neuronal intrinsic properties in brain tissue slices at neuroprotective concentrations. Collectively, our findings provide insight into the role of syntaxin in neuronal cell death and validate an important target for neuroprotection.

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C-Derived Protein NS5A Targeting Kv2.1 Potassium Channels.

We present the design of an innovative molecular neuroprotective strategy and provide proof-of-concept for its implementation, relying on the injury-mediated activation of an ectopic gene construct. As oxidative injury leads to the intracellular liberation of zinc, we hypothesize that tapping onto the zinc-activated metal regulatory element (MRE) transcription factor 1 system to drive expression of the Kv2.1-targeted hepatitis C protein NS5A (hepatitis C nonstructural protein 5A) will provide neuroprotection by preventing cell death-enabling cellular potassium loss in rat cortical neurons in vitro. Indeed, using biochemical and morphologic assays, we demonstrate rapid expression of MRE-driven products in neurons. Further, we report that MRE-driven NS5A expression, induced by a slowly evolving excitotoxic stimulus, functionally blocks injurious, enhanced Kv2.1 potassium whole-cell currents and improves neuronal viability. We suggest this form of "on-demand" neuroprotection could provide the basis for a tenable therapeutic strategy to prevent neuronal cell death in neurodegeneration.

Imprecision in Precision Medicine: Differential Response of a Disease-Linked GluN2A Mutant to NMDA Channel Blockers.

Mutations in N-methyl-d-aspartate receptors (NMDAR) subunits have been implicated in a growing number of human neurodevelopmental disorders. Previously, a de novo mutation in GRIN2A, encoding the GluN2A subunit, was identified in a patient with severe epilepsy and developmental delay. This missense mutation, which leads to GluN2A-P552R, produces significant dendrotoxicity in transfected rodent cortical neurons, as evidenced by pronounced dendritic blebbing. This injurious process can be prevented by treatment with the NMDA antagonist memantine. Given the increasing use of FDA approved NMDA antagonists to treat patients with GRIN mutations, who may have seizures refractory to traditional anti-epileptic drugs, we investigated whether additional NMDA antagonists were effective in attenuating neurotoxicity associated with GluN2A-P552R expression. Intriguingly, we found that while treatment with memantine can effectively block GluN2A-P552R-mediated dendrotoxicity, treatment with ketamine does not, despite the fact that both drugs work as open NMDAR channel blockers. Interestingly, we found that neurons expressing GluN2A-P552R were more vulnerable to an excitotoxic insult-an effect that, in this case, could be equally rescued by both memantine and ketamine. These findings suggest that GluN2A-P552R induced dendrotoxicity and increased vulnerability to excitotoxic stress are mediated through two distinct mechanisms. The differences between memantine and ketamine in halting GluN2A-P552R dendrotoxicity could not be explained by NMDA antagonist induced changes in MAP or Src kinase activation, previously shown to participate in NMDA-induced excitotoxicity. Our findings strongly suggest that not all NMDA antagonists may be of equal clinical utility in treating GRIN2A-mediated neurological disorders, despite a shared mechanism of action.